Enzyme Polymorphism in Sitophilus oryzae (Linnaeus, 1763) and Sitophilus zeamais (Motschulsky, 1855) (Coleoptera, Curculionidae) in southern Brazil / Polimorfismo Enzimático em Sitophilus oryzae (Linnaeus, 1763) e Sitophilus zeamais (Motschulsky, 1855) (Coleoptera, Curculionidae) no sul do Brasil

Beetles of the species Sitophilus oryzae and S. zeamais are pests of great economic importance since they attack not only rice and maize but also several other cereals. In fact, these beetles are one of the most visible threats to sustainable food production. Current study estimated the genetic variability of S. oryzae in two samples, one from the State of Paraná (PR), Brazil, and another from the state of Rio Grande do Sul (RS), Brazil, and a sample of S. zeamais from the State of Santa Catarina (SC), Brazil. Isozyme electrophoresis in starch gel technique was employed to analyze eight enzyme systems (AAT, ACP, GDH, GPI, IDH, MDH, PGM and ME). Average heterozygosity rates were 0.0091, 0.0100 and 0.0000 and expected heterozygosity rates were 0.0419, 0.0452 and 0.0000 respectively for the samples of PR, SC and RS samples. The percentage of polymorphic loci was 30% in the PR sample, 0% in the RS sample and 30% in the SC sample. Genetic identity rates were I=0.9983 between samples from PR and RS; I = 0.6892 between PR and SC, and I = 0.6925 between SC and RS. Nei´s (1978) genetic distance rates were 0.0017, 0.3722 and 0.3675. Samples presented low genetic variability.

Os besouros Sitophilus oryzae e S. zeamais são considerados pragas de grande importância econômica. Além do arroz e do milho, eles atacam outros diversos cereais. São uma das ameaças mais visíveis para a produção sustentável de alimentos. Este trabalho teve como objetivo estimar a variabilidade genética de S. oryzae em duas amostras, uma do Estado do Paraná (PR), e outra do Rio Grande do Sul (RS) e uma amostra de S. zeamais de Santa Catarina (SC). Utilizou-se a técnica de eletroforese de isozimas em gel de amido para a análise de oito sistemas enzimáticos (AAT, ACP, GDH, GPI, IDH, MDH, ME e PGM). A heterozigosidade média observada foi de 0,0091, 0,0100 e 0,0000 e a esperada foi de 0,0419, 0,0452 e 0,0000 para as amostras do PR, SC e RS, respectivamente. A porcentagem de locos polimórficos foi de 30, 0 e 30% nas amostras do PR, RS e SC, respectivamente. Os valores para identidade genética foram de I = 0,9983 entre as amostras do PR e RS; I = 0,6892 entre PR e SC e I = 0,6925 entre SC e RS, e os valores da distância genética de Nei (1978) foram 0,0017, 0,3722 e 0,3675, respectivamente. As amostras apresentaram pouca variabilidade genética.

Transcriptome responses to heat- and cold-stress in ladybirds (Cryptolaemus montrouzieri Mulasnt) analyzed by deep-sequencing

BACKGROUND: Changed temperature not only threaten agricultural production, but they also affect individual biological behavior, population and community of many insects, and consequently reduce the stability of our ecosystem. Insect's ability to respond to temperature stress evolved through a complex adaptive process, thus resulting in varied temperature tolerance among different insects. Both high and low extreme temperatures are detrimental to insect development since they constitute an important abiotic stress capable of inducing abnormal biological responses. Many studies on heat or cold tolerance of ladybirds have focused on measurements of physiological and biochemical indexes such as supercooling point, higher/lower lethal temperatures, survival rate, dry body weight, water content, and developmental duration. And studies of the molecular mechanisms of ladybird responses to heat or cold stress have focused on single genes, such as those encoding heat shock proteins, but has not been analyzed by transcriptome profiling. RESULTS: In this study, we report the use of Digital Gene Expression (DGE) tag profiling to gain insight into transcriptional events associated with heat- and cold-stress in C. montrouzieri. About 6 million tags (49 bp in length) were sequenced in a heat stress group, a cold stress group and a negative control group. We obtained 687 and 573 genes that showed significantly altered expression levels following heat and cold shock treatments, respectively. Analysis of the global gene expression pattern suggested that 42 enzyme-encoding genes mapped to many Gene Ontology terms are associated with insect's response to heat- and cold-stress. CONCLUSIONS: These results provide a global assessment of genes and molecular mechanisms involved in heat and cold tolerance.

New morphological aspects and phylogenetic considerations of Cicindis Bruch (Coleoptera: Carabidae: Cicindini)

Cicindis Bruch is a monospecific genus of carabid beetles endemic to Argentina. In this contribution, Cicindis horni Bruch is re-described, with addition of new morphological features of male internal sac, female genital tract and elytral closure. New information on the species' habitat and distribution is also provided. The phylogenetic placement and relationships of Cicindis within the family Carabidae are discussed on the basis of a cladistic analysis. Terminal taxa included representatives of all subfamilies of Carabidae and supertribes of Carabinae, with a major sampling of those taxa considered to be closely related to Cicindini by previous authors. The phylogenetic analysis shows the basal position of Cicindis in a clade that includes Ozaeninae, Omophronini, Scaritinae and Conjuncta. A close relationship of Cicindis with Ozaenini + Metriini is supported by the particular closure of the procoxa and the ventral position of the oviduct with respect to the spermatheca.

Phylogenetic relationships of the genus Hoplia Illiger (Scarabaeidae: Hopliinae)

Results of phylogenetic analysis based on 34 morphological characters of 24 species of 11 genera of Hopliinae from Europe, Japan, South Africa, Madagascar, North and Central America, indicates that the genus Hoplia is a monophyletic group with species distributed in Europe, Japan and America. Based in this analysis the Asiatic genus Ectinohoplia is the closest relative of the genus Hoplia, and the South American genus Barybas (Melolonthinae: Macrodactylini) is the sister group of Hopliinae.

Comparison of mtDNA extracting methods for common sarcosaphagous insects / 法医学杂志

OBJECTIVE@#To compare effects of three different methods for mtDNA extraction from common sarcosaphagous insects including cetyl trimethyl ammonium bromide (CTAB) method, sodium dodecyl sulfate-potassium acetate (SDS-KAc) method and sodium dodecyl sulfate-proteinase K (SDS-PK) method.@*METHODS@#Seventy-two insects from four species [Chrysomya megacephala (Fabricius, 1784), Eusilpha bicolor (Fairmaire, 1896), Paraeutrichopus pecoudi (Mateu, 1954), Vespa velutina (Lepeletier, 1836)] were collected from the corpses of the rabbits in Changsha district. The total DNA of above samples was extracted by CTAB, SDS-Kac and SDS-PK methods. The purity and concentration of DNA were examined by protein-nucleic acid spectrophotometry, and mtDNA were amplified by specific primers and PCR products were detected by agarose gel electrophoresis. Then PCR products were sequenced and subsequently up-loaded to GenBank.@*RESULTS@#mtDNA was successfully extracted with three methods from most of the samples. The SDS-PK method was better in DNA purity compared to other methods and the CTAB method was superior in extracting DNA from old samples, while SDS-KAc method showed no significant difference for extraction effects of different samples.@*CONCLUSION@#The most appropriate method should be chosen depending on different situations. SDS-PK method is expected to obtain high-quality DNA, while CTAB method is preferred in extracting obsolete samples. SDS-KAc method is low cost and can be used in various kinds of preliminary experiments.

Comparison of mtDNA extraction from different parts of sarcosaphagous insects / 法医学杂志

OBJECTIVE@#To explore mitochondrial DNA (mtDNA) extraction effects of different parts from sarcosaphagous insects using improved cetyltriethylammnonium bromide (CTAB) method.@*METHODS@#Thirteen Lucilia sericata (Meigen) and 13 Nicrophorus fossor (Erichson) were collected from the corpses of rabbits placed on the outdoor lawn in Huhehot district. Four parts (head, chest muscle, legs and wings) of insect were collected, and the mtDNA of all samples were extracted using CTAB method. The purity and concentration were tested using protein and nucleic acid spectrophotometry. The integrity of the extracted mtDNA and PCR products were checked by agarose gel electrophoresis. The PCR products were sequenced and the obtained sequences were imputed into GenBank for comparison.@*RESULTS@#mtDNA were successfully extracted from 10 head samples, 6 legs samples, 4 wing samples and 13 chest muscle samples of the Lucilia sericata (Meigen). Also, mtDNA were successfully extracted from 5 head samples, 8 legs samples, 3 wing samples and 13 chest muscle samples of the Nicrophorus fossor (Erichson).@*CONCLUSION@#mtDNA can be obtained from chest muscle and other parts of sarcosaphagous insects using the improved CTAB method.

Karyotype, heterochromatin distribution and meiosis of Zabrotes subfasciatus (Bohemann) (Coleoptera: Chrysomelidae, Bruchinae) / Cariótipo, distribuição da heterocromatina e meiose de Zabrotes subfasciatus (Bohemann) (Coleoptera: Chrysomelidae, Bruchinae)

Zabrotes subfasciatus (Boh.) é estudada intensivamente em termos agronômicos e bioquímicos por causar danos aos grãos de leguminosas armazenados. No entanto, os dados publicados sobre o seu cariótipo são escassos e conflitantes. Assim, o objetivo deste estudo foi descrever o cariótipo e a meiose desse inseto e analisar o padrão de bandas-C de seus cromossomos. Foram analisados os gânglios cerebrais de pré-pupas e os testículos de adultos e pupas com adaptação de uma técnica que permitiu boa qualidade de preparo dos cromossomos dessa espécie. Todos os indivíduos apresentaram 26 cromossomos nas metáfases mitóticas. Esses cromossomos foram classificados em: acrocêntricos (cromossomo X); submetacêntricos (pares 4 e 5); subtelocêntricos (par 12 e cromossomo y); metacêntricos (demais pares). O cromossomo 5 apresentou uma constrição secundária. Todos os cromossomos apresentaram heterocromatina próximo ao centrômero e os cromossomos 5, 9 e X, nos braços longos. O cromossomo X mostrou-se heteropicnótico durante toda a prófase da primeira divisão meiótica. As subfases da prófase I foram pouco distintas e a meiose II de difícil identificação. Os testículos de todos os machos apresentaram poucas células. Os bivalentes apresentaram a forma de bastão na metáfase I. O seu cariótipo constitui-se de 26 cromossomos, sendo as fórmulas cariotípicas 2n = 24 + XX nas fêmeas e 2n = 24 + Xyp e n = 12 + X ou n = 12 + y nos machos.

Zabrotes subfasciatus (Boh.) has been extensively studied in its agronomic and biochemical aspects due to its importance as a damaging insect to leguminous grains during storage. The few cytogenetic studies published on this species yielded conflicting results. In this study, the karyotype was analyzed in order to accurately describe the chromosome C-banding patterns and meiosis. The brain ganglion at the prepupa and the adult and pupal testes were analyzed. All individuals had 26 chromosomes in both brain ganglion and spermatogonic mitotic metaphases. These chromosomes were classified as follows: the 12th pair and the Y chromosome were telocentric; the X chromosome was acrocentric; the 4th and 5th pairs were submetacentric; and the remaining pairs were all metacentric. One of the members of the 5th pair presented a secondary constriction. All chromosomes presented pericentromeric heterochromatin. The large arms of the pairs 5, 9 and X presented heterochromatin. The X chromosome showed to be heteropyknotic throughout the prophase of the first meiotic division. The subphases of prophase I were atypical and meiosis II was rarely identified. Testes of all males showed a few cells; the bivalents were rod-like shaped in metaphase I. Karyological formulae were 2n = 24 + XX in females and 2n = 24 + XYp and either n = 12 + X or n = 12 + Y in males.

The role of stress proteins in responses of a montane willow leaf beetle to environmental temperature variation.

The heat shock response is a critical mechanism by which organisms buffer effects of variable and unpredictable environmental temperatures. Upregulation of heat shock proteins (Hsps) increases survival after exposure to stressful conditions in nature, although benefits of Hsp expression are often balanced by costs to growth and reproductive success. Hsp-assisted folding of variant polypeptides may prevent development of unfit phenotypes; thus, some differences in Hsp expression among natural populations of ectotherms may be due to interactions between enzyme variants (allozymes) and Hsps. In the Sierra willow leaf beetle Chrysomela aeneicollis, which lives in highly variable thermal habitats at the southern edge of their range in the Eastern Sierra Nevada, California, allele frequencies at the enzyme locus phosphoglucose isomerase (PGI) vary across a climatic latitudinal gradient. PGI allozymes differ in kinetic properties,and expression of a 70 kDa Hsp differs between populations, along elevation gradients,and among PGI genotypes. Differences in Hsp70 expression among PGI genotypes correspond to differences in thermal tolerance and traits important for reproductive success, such as running speed, survival and fecundity. Thus, differential Hsp expression among genotypes may allow functionally important genetic variation to persist, allowing populations to respond effectively to environmental change.

Evaluation of hot saline solution and restriction endonuclease techniques in cytogenetic studies of Cycloneda sanguinea L. (Coccinellidae)

The goal of the present study was to determine if simple methods, especially hot saline solution (HSS) and MspI and HaeIII restriction endonucleases, which do not require special equipments, may be helpful in studies of genetic variability in the lady beetle, Cycloneda sanguinea. The HSS method extracted the heterochromatin region, suggesting that it is composed mostly of DNA rich in A-T base pairs. However, the X and y chromosomes were resistant to HSS banding. These bands facilitated the identification of each chromosome. In this study, we used the restriction endonucleases with different G-C base target sequences: MspI C/GGC and HaeIII GG/CC. The use of restriction enzyme MspI did not show an effect on the autosomal chromosomes. On the other hand, the sex pair showed a pale staining, to help in the recognition of these chromosomes. HaeIII produced characteristic bands which were identified all along the chromosomes, facilitating the identification of each chromosome. Based on these results, we can consider the heterochromatin being heterogeneous. The findings obtained here, using different chromosomal banding techniques, may be useful in the identification of intraspecific chomosome variability, specifically in Coccinellidae (Coleoptera) chromosomes, even without special equipment.

Taxonomy and phylogeny of a new Central American beetle genus: Catrachia (Coleoptera: Scarabaeidae)

A new genus and two species of Melolonthinae from Honduras and Nicaragua are described. A phylogenetic analysis, carried out using morphological characters, suggests that Catrachia is a strongly supported monophyletic group. Catrachia is therefore established as a new genus, constituted by two new species, Catrachia mariana and Catrachia nica.

En este trabajo se describe el género Catrachia y dos especies nuevas, Catrachia mariana y Catrachia nica de Honduras y Nicaragua respectivamente. En análisis filogenético basado en caracteres morfológicos demostró que Catrachia es un grupo monofilético fuertemente soportado lo cual permite su consideración como género nuevo.

Karyotype characterization, constitutive heterochromatin and nucleolus organizer regions of Paranaita opima (Coleoptera, Chrysomelidae, Alticinae)

Species of the subtribe Oedionychina not only have a highly uniform diploid number of 2n = 22 (20+X+y) but have the karyotypic peculiarity of possessing extremely large sex chromosomes. We analyzed Paranaita opima embryos and gonadal cells to determine their diploid number, chromosomal morphology, type of sex determination system, constitutive heterochromatin pattern and which chromosomes bear nucleolus organizer regions (NORs). The diploid number of P. opima was 2n = 22 (20+XY/XX) with all chromosomes being metacentric. Chromosome pair 6 showed an interstitial secondary constriction on the short arm. The C-banding technique revealed centromeric constitutive heterochromatin in all chromosomes, which, in pair 6, extended up to the secondary constriction of the short arm, additional C-bands also being present on the Y chromosome. Silver nitrate nucleolar organizer region (Ag-NOR) staining showed NORs on the secondary constriction of pair 6. Fluorochrome analysis with chromomycin A3 (CMA3), 4'-6-diamidino-2-phenylindole (DAPI) and the distamycin A (DA) counterstain showed that the short arm of chromosome pair 6 exhibited a GC-rich block extending from the proximal to the median region, including part of the secondary constriction. The same techniques also showed AT-rich blocks at the centromeric region of all chromosomes and at the terminal region of the short arm of pair 6. The basic karyotype characteristics and C band pattern of P. opima are similar to those described for other species in the subtribe Oedionychina. The pattern of autosomal NORs observed in P. opima corresponds to that registered in the majority of the Chrysomelidae species.

Chromosome evolution in tiger beetles: Karyotypes and localization of 18S rDNA loci in Neotropical Megacephalini (Coleoptera, Cicindelidae)

Four Neotropical tiger beetle species, three from the genus Megacephala and one from the genus Oxycheila, currently assigned to the tribe Megacephalini were examined cytogenetically. All three Megacephala species showed simple sex chromosome systems of the X0/XX type but different numbers of autosomal pairs (15 in M. cruciata, 14 in M. sobrina and 12 in M. rutilans), while Oxycheila tristis was inferred to have a multiple sex chromosome system with four X chromosomes (2n = 24 + X1X2X3X4Y/X1X1X2X2X3X3X4X4). Fluorescence in situ hybridization (FISH) using a PCR-amplified 18S rDNA fragment as a probe revealed the presence of rDNA clusters located exclusively on the autosomes in all the Megacephala species (five clusters in M. cruciata, eight in M. sobrina and three in M. rutilans), indicating variability in the number of clusters and the presence of structural polymorphisms. The same methodology showed that O. tristis had six rDNA clusters, apparently also located on the autosomes. Although our data also show cytogenetic variability within the genus Megacephala, our findings support the most accepted hypothesis for chromosome evolution in the family Cicindelidae. The description of multiple sex chromosomes in O. tristis along with phylogenetic analyses and larval morphological characters may be assumed as an additional evidence for the exclusion of the genus Oxycheila and related taxa from the tribe Megacephalini.

Karyotype, C-and fluorescence banding pattern, NOR location and FISH study of five Scarabaeidae (Coleoptera) species

Meiotic chromosomes obtained from members of the coleopteran subfamilies Rutelinae and Dynastinae were studied using standard and silver nitrate staining, C-banding, base-specific fluorochromes and fluorescent in situ hybridization (FISH). The study presents detailed karyotipic descripitions of three Rutelinae species (Geniates borelli, Macraspis festiva and Pelidnota pallidipennis), and two Dynastinae species (Lygirus ebenus and Strategus surinamensis hirtus) with special emphasis on the distribution and variability of constitutive heterochromatin and the nucleolar organizer region (NOR). We found that for G. borelli, P. pallidipennis, L. ebenus and S. s hirtus the karyotype was 2n = 20 (9II + Xy p), with G. borelli, P. pallidipennis and L. ebenus showed meta-submetacentric chromosomes which gradually decreased in size. For Macraspis festiva the karyotype was 2n = 18 (8II + Xy p). In L. ebenus we found that the NOR was located on an autosome, but in the other four species it occurred on the sex bivalents. In all five species the constitutive heterochromatin (CH) was predominantly pericentromeric while the X chromosomes were almost completely heterochomatic, although CMA3/DA/DAPI staining showed intra and interspecific variation in the bright fluorescence of the constitutive heterochromatin. The FISH technique showed rDNA sites on the X chromosome of the Rutelinae species.

Phylogenetic relationships and the evolution of mimicry in the Chauliognathus yellow-black species complex (Coleoptera: Cantharidae) inferred from mitochondrial COI sequences

The phylogenetic relationships of twelve species of Chauliognathus were investigated by studying the mitochondrial cytochrome oxidase I gene. A 678 bp fragment of the COI gene was sequenced to test the hypothesis that the Müllerian mimicry species of the "yellow-black" complex make up a monophyletic clade, separated from species with other colour patterns. The data set was analysed by neighbour-joining, maximum parsimony and maximum likelihood procedures. The results support a single origin of the yellow-black colour pattern during the evolution of the genus, with one main clade formed by Chauliognathus lineatus, C. tetrapunctatus, C. riograndensis, C. flavipes, C. octomaculatus, C. fallax, and another one formed by two species, C. expansus and C sp 1, plus an orange-black-coloured species. The nucleotide divergences found between C. sp 3 (black) and the other species studied fall within the level expected for species from different genera. The similarity of colour patterns of the yellow-black species has been considered an example of Müllerian mimicry by conservation of the ancestral state with some minor modifications.

Elytra colour polymorphism and randomness of matings in Chauliognathus fallax Germar 1824 from southern Brazil (Coleoptera, Cantharidae)

Beetles of the species Chauliognathus fallax Germar 1824 are polymorphic for elytron colouration with six morphs distinguished on the basis of black pigmentation on a yellow background. We investigated samples of C. fallax taken in eight consecutive weeks aiming to determine the frequency of the morphs which were grouped, for statistical analysis, into three classes according to elytra pigmentation as well as the possible occurrence of assortative mating. Our results showed a variation in the frequency of the classes throughout the season, both in males and females, with the maximum frequency of each class at the fourth and fifth week. The three classes (A, B, C) showed the same pattern of variation, and class B was always the more frequent. To test randomness of matings two approaches were taken: in the first, we compared the frequency of each class in copulating and non-copulating insects. In the second, the frequency of each class in the whole sample was taken as the probability of occurrence of the respective class; then, using the criterion of the probability of independent events we calculated the expected proportion of copulating insects for each pair of events. Both methods gave non-significant differences, suggesting that the matings were random.